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WO2023125704A1 - 一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途 - Google Patents

一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途 Download PDF

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WO2023125704A1
WO2023125704A1 PCT/CN2022/142961 CN2022142961W WO2023125704A1 WO 2023125704 A1 WO2023125704 A1 WO 2023125704A1 CN 2022142961 W CN2022142961 W CN 2022142961W WO 2023125704 A1 WO2023125704 A1 WO 2023125704A1
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activated
lymphocytes
cell
expanded
serum
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PCT/CN2022/142961
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French (fr)
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王歈
邓声菊
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北京永泰生物制品有限公司
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Definitions

  • the invention relates to the field of biomedicine, in particular to a method for efficiently expanding and activating lymphocytes and its use in preventing and treating neurological diseases.
  • Cerebral stroke is an acute cerebrovascular disease, ischemic damage to brain tissue caused by the sudden rupture of blood vessels in the brain or the inability of blood to flow into the brain due to vascular obstruction, and has high morbidity, high mortality and fatality. Features such as high residual rate. Occlusion and stenosis of the internal carotid artery and vertebral artery can cause ischemic stroke, and severe cases can cause death.
  • Traumatic brain injury is mostly traumatic brain injury caused by foreign objects, and often causes permanent dysfunction of varying degrees. Brain lesions in different regions can cause focal symptoms including abnormalities in motor, sensory, speech, visual, auditory and other regions. Diffuse brain damage can even affect memory, sleep or cause confusion and coma.
  • Neurodegenerative diseases are chronic progressive degeneration of central nervous tissue due to loss of neurons or glial cells in the brain or spinal cord, including Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis syndrome, multiple sclerosis, etc.
  • Parkinson's disease Alzheimer's disease
  • Huntington's disease Huntington's disease
  • amyotrophic lateral sclerosis syndrome multiple sclerosis, etc.
  • Ataxia is clumsy and uncoordinated movement resulting from cerebellar, proprioceptive, and vestibular dysfunction, which can cause disturbances in balance, posture, gait, and speech when the trunk, extremities, and throat muscles are involved.
  • Ataxia is a complex manifestation of multiple disorders and requires investigation of its underlying etiology.
  • Cerebellar ataxia is a common presentation in neurological patients with various etiologies. Cerebellar degeneration can be long and slowly progressive, but acute cerebellar swelling from infarction, edema, or hemorrhage can have rapid and severe adverse effects and is a neurological emergency.
  • the etiology of ataxia is complex, and the development of molecular, genetic, and imaging diagnostic techniques provides more evidence for the correct diagnosis of various types of ataxia.
  • the pathogenesis of the patient has not been clarified. Due to the influence of individual differences in patients, clinical drug treatment methods lack sufficient evidence-based medical basis, and the curative effect is also difficult to be sure. Research and development of safer and more effective ataxia drugs to meet unmet clinical needs.
  • Peripheral nerve injury is a common clinical disease mainly caused by factors such as trauma, tumors, and metabolic diseases. It often leads to partial or total loss of motor, sensory, and autonomic functions of the affected segment of the body, as well as intractable neuralgia, which seriously affects patients. quality of life and impose a heavy burden on families and society.
  • the cell types used for peripheral nerve injury repair mainly include mature somatic cells such as Schwann cells and olfactory ensheathing cells, stem cells such as embryonic stem cells, neural stem cells, mesenchymal stem cells, myoblasts and induced pluripotent stem cells.
  • mature somatic cells such as Schwann cells and olfactory ensheathing cells
  • stem cells such as embryonic stem cells, neural stem cells, mesenchymal stem cells, myoblasts and induced pluripotent stem cells.
  • Diabetic patients tend to have weak immunity and are prone to various diseases: diabetic patients have disordered glucose metabolism, high blood sugar, increased sugar content in tissue cells, and accumulation of pyruvate, which is conducive to the growth and reproduction of bacteria; fat metabolism disorders, increased triglycerides, It provides rich nutrition for bacteria; the formation of albumin decreases, the level of antibodies and complements in the body decreases, immunity decreases, blood viscosity increases, and microcirculation disorders affect the oxygen exchange between blood and tissues, resulting in decreased body resistance. Metabolic disorders in diabetic patients lead to liver damage, decreased function of transforming vitamin A, resulting in damage to the integrity of respiratory mucosal epithelium, decreased defense function, and susceptibility to bacterial infection.
  • Diabetic patients or their relatives are often accompanied by other autoimmune diseases, such as diffuse goiter with hyperthyroidism, chronic lymphatic thyroiditis, myasthenia gravis, pernicious anemia.
  • autoimmune diseases such as diffuse goiter with hyperthyroidism, chronic lymphatic thyroiditis, myasthenia gravis, pernicious anemia.
  • In elderly patients with type II diabetes the incidence of malignant tumors is significantly increased. Insulin resistance in patients can lead to hyperinsulinemia, and insulin can promote the growth of malignant tumors.
  • Allergic rhinitis also known as allergic rhinitis, Anaphylactic Rhinitis, AR is a non-infectious inflammatory disease of the nasal mucosa.
  • Eczema is a common allergic disease, prone to repeated attacks, and the patients are regardless of age, gender and region.
  • the causes of eczema may be related to the internal factors of the body, external environmental factors, and social and psychological factors.
  • Part of the immune cells in the human body are fighting soldiers with a feverish mind, regardless of whether they are the enemy or friend, and unconventional weapons such as antibodies and complements open fire and start attacking their own people.
  • Urticaria is commonly called as wheal rash.
  • a localized edema reaction caused by dilation and increased permeability of small blood vessels in the skin and mucous membranes, which usually subsides within 2 to 24 hours, but new rashes occur repeatedly. The course of disease protracted for several days to several months. Clinically more common.
  • Menopausal syndrome is a transitional period in which women's ovarian function gradually declines to disappear completely.
  • a series of clinical symptoms appear due to physiological and psychological changes, such as hot sweating, irritability, palpitations, insomnia, or depression and forgetfulness.
  • this disease belongs to the category of "all syndromes before and after menopause" in traditional Chinese medicine.
  • Stem cells can directionally differentiate enough egg cells, replenish the number of egg cells, stimulate the secretion of estrogen, and maintain the normal shape and function of the ovary.
  • stem cells to treat ovarian aging can delay the arrival of women's menopause, delay menopause, increase the psychological age, target effect, stimulate the secretion of estrogen hormones in the body, increase the level of estrogen in the body, and improve many problems of women's menopause. Delay the onset of menopause.
  • the expanded activated lymphocytes (Expanded Activated Lymphocytes) in serum-free cell culture are prepared from mononuclear cells in the patient's own peripheral blood. Lymphocytes are the main functional cells, and the average number of CD8+ T cells in a single reinfusion of patients is 4.70 ⁇ 1.47 ⁇ 10 9 , which has shown good therapeutic activity in clinical practice.
  • the prior art discloses methods for culturing and expanding activated lymphocytes without serum, but it is necessary to optimize the extraction and separation of peripheral blood mononuclear cells (PBMC), the types and ratios of lymphocyte activators, and serum-free medium to improve this method.
  • PBMC peripheral blood mononuclear cells
  • the lymphocyte expansion multiple, cell viability and biological activity obtained by the method meet the clinical needs of stable and controllable cell quality and safe and effective treatment.
  • the object of the present invention is to provide a method for preparing a stock solution of activated and expanded lymphocytes, comprising the following steps:
  • PBMC peripheral blood mononuclear cells
  • the lymphocyte activator is selected from any one of anti-human CD2 antibody, anti-human CD3 antibody, anti-human CD28 antibody, phytohemagglutinin (PHA) or a combination thereof or an antibody-containing carrier immobilized on the carrier, said
  • the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof;
  • step (2) Place the cultured activated lymphocytes prepared in step (1) in a serum-free medium according to the cell density of (0.5-5) ⁇ 106 /ml, and carry out subculture to obtain an activated lymphocyte culture,
  • the subculture temperature is 37.0°C ⁇ 1.0°C
  • the activation culture environment contains 7.5% ⁇ 1.0% CO 2
  • the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof, and the number of subculture generations For 1-5 generations;
  • step (3) Add serum-free medium 5-10 times its volume to the activated lymphocyte culture obtained in step (2) for subculture, and carry out expansion culture, wherein, the expansion culture temperature is 37.0 ° C ⁇ 1.0 ° C, the activation
  • the culture environment contains 7.5% ⁇ 1.0% CO 2
  • the serum-free medium is selected from any one of KBM 581, GT-T551H3 or a combination thereof, and the number of expansion cultures is 1-5;
  • the initial density of the biological samples cultured and activated in the serum-free medium is (0.2-1) ⁇ 10 6 samples/ml.
  • the lymphocyte activator is an anti-human CD3 antibody, preferably 2.5 ⁇ g/ml-5 ⁇ g/ml, with a volume of 8-15 ml, more preferably 2.5 ⁇ g/ml-3.8 ⁇ g/ml , with a volume of 10-13ml.
  • the method for centrifuging whole blood to extract peripheral blood mononuclear cells comprises the following steps: adding a separation medium and a diluent to the anticoagulated whole blood, stirring, mixing, adding it to In the separation liquid, centrifuge at 1000-3000rpm*10-40min, collect the cell layer between the interface, add washing liquid, centrifuge, wash, collect the cells, and obtain that, wherein the separation medium is selected from hydroxyethyl starch 40 sodium chloride Any one of injection (HES), Percoll, Ficoll-Paque PLUS or a combination thereof, the volume ratio of whole blood: diluent is 1:1-2, and the diluent is selected from sodium chloride injection, Hank's buffer , Lactated Ringer's solution, any one of Dulbecco's phosphate buffer or a combination thereof, the separating liquid is selected from Hetastarch 40 Sodium Chloride Injection (HES), Ficoll, Lymphoprep,
  • HES Sodium Chloride In
  • the centrifugal condition is (1500-2500rpm)*(15-30min), preferably (2000-2500rpm)*(20-25min).
  • washing is centrifugal washing, and washing condition is (500-2000rpm)*(5-20min), centrifugal washing 1-5 time, is preferably (1000-1800rpm)*(10-15min), centrifugal Wash 2-3 times.
  • the cell density of the subculture in step (2) is (1-4) ⁇ 10 6 cells/ml
  • the pH of the subculture system after adding serum-free medium is 7.00-7.80.
  • step (2) the cell density of the subculture is (2-3) ⁇ 10 6 cells/ml, and the pH of the subculture system after adding serum-free medium is 7.02-7.76.
  • the osmotic pressure of the separation liquid is 310-350 mOsmol/kg, preferably 316-347 mOsmol/kg.
  • the number of subcultures is 2-3 generations.
  • step (3) when the subculture cell density increases to 1-10 times of the culture activated cell density, the expansion culture is started, and the lymphocyte culture obtained in step (2) subculture is added to Its volume is 6-8 times in total in serum-free medium for expansion culture, and the pH of the expansion culture system is 6.80-7.80.
  • step (3) when the subcultured cell density increases to 1.2-3 times of the cultured activated cell density, the expanded culture is started, and the pH of the expanded culture system is 6.88-7.70.
  • the number of expansion culture generations is 2-3 generations.
  • the centrifugal condition in step (4) is (1000-3000rpm)*(1-10min), preferably (1500-2800rpm)*(2-8min), more preferably (2000-2500rpm )*(5-6min).
  • the cytokine IL-2 300-600IU/ml is optionally added in the serum-free medium, preferably 400-500IU/ml.
  • the pH in the serum-free medium is 6.9-7.9, preferably 7.2-7.4.
  • the culture equipment is selected from any one of incubators, shakers, and bioreactors.
  • the expansion factor of activated and expanded lymphocytes is ⁇ 900 times, preferably ⁇ 1000 times, more preferably ⁇ 1100 times.
  • the viability of activated and expanded lymphocytes is ⁇ 95%, preferably ⁇ 98%.
  • the number of CD8+ T cells in the activated and expanded lymphocytes is ⁇ 1 ⁇ 10 9 , preferably 1 ⁇ 10 9 -2 ⁇ 10 10 , more preferably 4-9.5 ⁇ 10 9 .
  • the biological activity of activated and expanded lymphocytes is KT 50 ⁇ 8.5, preferably KT 50 ⁇ 4, more preferably KT 50 ⁇ 0.7141.
  • Another object of the present invention is to provide a pharmaceutical composition containing the stock solution of activated and expanded lymphocytes, the composition is composed of (1-20) ⁇ 10 7 activated and expanded lymphocytes/ml, Composed of human serum albumin and physiological saline for injection, the viability of the activated and expanded lymphocytes is ⁇ 95%, wherein the CD8+ T cells in the activated and expanded lymphocytes are (1-20) ⁇ 10 7 /ml, the The pharmaceutical composition does not contain preservatives and antibiotics.
  • the composition is composed of (2-18) ⁇ 10 9 /ml activated and expanded lymphocyte concentration, 1-1.5% human serum albumin concentration and physiological saline for injection, and the activated and expanded lymphocyte concentration is 1-1.5%.
  • the viability of increased lymphocytes is ⁇ 98%, and the number of CD8+T cells in the activated and expanded lymphocytes is 1 ⁇ 10 9 -2 ⁇ 10 10 .
  • the cell amplification factor of the biological sample is ⁇ 900 times, preferably ⁇ 1000 times, more preferably ⁇ 1100 times.
  • the biological activity of activated and expanded lymphocytes is KT50 ⁇ 8.5, preferably KT50 ⁇ 4, more preferably KT50 ⁇ 0.7141.
  • the total number of activated and expanded lymphocytes in a single reinfusion of the patient is ⁇ 2 ⁇ 10 10 , and the cell viability within the validity period is ⁇ 85%.
  • the activity rate of activated and expanded lymphocytes of the composition within 12 hours of storage at 15-25°C is ⁇ 85%.
  • Another object of the present invention is to provide a method for preparing a pharmaceutical composition containing a stock solution of activated and expanded lymphocytes.
  • the stock solution of activated and expanded lymphocytes is resuspended in physiological saline for injection containing human albumin.
  • Another object of the present invention is to provide the method for activating and expanding lymphocytes of the present invention for immunotherapy, comprising the following steps:
  • Another object of the present invention is to provide the dosage regimen of activated and expanded lymphocytes of the present invention for immunotherapy, including the following regimens:
  • the patient receives 1-5 times of intravenous infusion, once every two weeks;
  • the number of activated and expanded lymphocytes in each intravenous infusion is ⁇ 5 ⁇ 10 8 lymphocytes, preferably ⁇ (1-20) ⁇ 10 9 lymphocytes.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes for the preparation of drugs for the prevention and treatment of patients with neurological diseases who need to enhance immunity, nerve repair and/or neurotrophy to repair and/or Or trophic nerve trauma, nerve damage caused by ischemia and hypoxia.
  • the neurological disease is selected from ataxia, dementia, epilepsy, stroke, sequelae of stroke, Parkinson's disease, neurodegeneration, hand tremor, peripheral nerve and muscle disease , headache, sleep disturbance, dizziness, any of nervous system inflammation or its complications.
  • the ataxia is selected from any one of cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia or a combination thereof.
  • the ataxia is selected from any one of acquired ataxia, hereditary ataxia and non-hereditary degenerative ataxia.
  • Another object of the present invention is to provide the combination of activated and expanded lymphocytes and rehabilitation training for the prevention and treatment of neurological diseases.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes in the preparation of medicaments for treating patients with allergic diseases.
  • the allergic disease includes any one of rhinitis, eczema, and urticaria.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes in the preparation of medicaments for treating menopausal syndrome patients.
  • Another object of the present invention is to provide the application of activated and expanded lymphocytes in the preparation of medicaments for treating diabetic patients.
  • the present invention uses the biological activity of expanded and activated lymphocytes (RTCA, taking the effect-to-target ratio of 40:1 as an example) to characterize the therapeutic activity of activated and expanded lymphocytes.
  • RTCA biological activity of expanded and activated lymphocytes
  • effector cells i.e., expanded and activated lymphocytes
  • a specific density 50 ⁇ l per well with a density of 1.6 ⁇ 10 7 cell/ml
  • real-time , dynamic and quantitative tracking of cell morphology, proliferation and differentiation data that is, the change of electrical impedance produced by the contact of living cells with the microelectrodes in the detection plate.
  • the instrument-specific software converts the resistance signal into a specific cell index, and the software monitors the generated cell index curve in real time to show the state of cell adhesion, stretching, growth, death, etc. in different time periods.
  • target cells such as HepG2 cells
  • KT 50 i.e., the time for effector cells to kill 50% of target cells
  • KT 50 value i.e. activated Expanded lymphocytes
  • said percentage is volume/volume percentage;
  • said percentage is volume/weight percentage;
  • said percentages are weight/volume percentages; the remainder are weight/weight percentages.
  • the present invention has the following beneficial technical effects:
  • the activated and expanded lymphocytes prepared by the present invention have the advantages of high cell expansion efficiency, high cell viability, stable cell activity, long validity period, safety and effectiveness, and small side effects, effectively solving the problem of individualized immune cell therapy.
  • the key issues are: one is to use a serum-free culture system with stable cell culture efficiency, and continue to culture for 12 days to maintain the killing activity and expansion ability of the cells, and to avoid the possible contamination of pathogenic microorganisms and other effects on cell growth caused by the use of serum
  • the cell expansion efficiency is high, there is no need to use a mononuclear cell collection machine, and the high-efficiency amplification is more than 1000 times, which avoids the need for traditional immune cell therapy to use a mononuclear cell collection machine to collect a large number of initial peripheral blood mononuclear cells for patients.
  • the third is that the cells have high activity and good stability, and the cells can be stored for 12 hours without affecting their therapeutic effect; the fourth is to solve the technical problems of the standardized quality control system and large-scale production of individual products, and obtain The technical process of cell products with relatively uniform quality can significantly improve the targeting and precision of treatment and the compliance of patients.
  • the activated and expanded lymphocytes prepared by the present invention are used to prevent and treat neurological disease patients who need to enhance immunity, nerve repair and/or neurotrophy to repair and/or nourish neurological trauma, deficient Nerve damage caused by blood hypoxia can significantly improve the patient's nerve conduction, joint flexibility and motor coordination, help improve and enhance the patient's body control and make their mind clearer, and significantly reduce dizziness and movement in patients with cerebellar ataxia Inconvenience and other symptoms can significantly improve the quality of life of patients and accelerate the recovery of patients. It has the advantages of high cell expansion efficiency, high cell viability, good cell stability, significant curative effect, fast recovery, safety and effectiveness, and small side effects. Targeting and precision of cell therapy, and significantly improve patient compliance.
  • the lymphocyte expansion method of the present invention has the advantages of simple operation, controllable quality, and suitability for industrial production.
  • PBMC peripheral blood mononuclear cells
  • Patient 1 Cerebellar ataxia, age: 52 years old, sex: male.
  • the serum-free cell culture medium is: GT-T551H3 serum-free cell culture medium (the concentration of IL-2 in the culture medium is 500IU/ml, pH 7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of 0.9% sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation is completed, pour off the supernatant, shake off the cell pellet, add 5ml of serum-free cell culture medium, mix well, take 10 ⁇ l of cell suspension for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.8 ⁇ 106 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody, samples were taken, sterility checked and labeled, and the Place in a cell culture incubator for cultivation.
  • the culture conditions in the incubator are: 37° C., 7.5% CO 2 .
  • the growth condition is good (see Figure 2), take a sample and carry out cell counting, when the viable cell density is not lower than 1.0 ⁇ 106 /ml, mix the cell culture medium with 760ml Serum-free cell culture medium, poured into the cell culture bag, and put it back into the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the cell stock solution at a concentration of 8 ⁇ 10 7 /ml in physiological saline for injection containing 1% human serum albumin.
  • Patient 2 cerebellar ataxia, age: 65 years old, sex: male.
  • the serum-free cell culture medium is: KBM 581 serum-free cell culture medium (the concentration of IL-2 in the culture medium is 500IU/ml, pH 7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation was completed, the supernatant was poured off, the cell pellet was shaken off, and 5ml of serum-free cell culture medium was added, after mixing, 10 ⁇ l of the cell suspension was taken for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.2 ⁇ 10 6 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody. A sample is taken, checked for sterility and labeled, and placed in a cell incubator for incubation.
  • the culture conditions in the incubator are: 37° C., 7.5% CO 2 .
  • the day of blood collection is considered as day 0, and the day after inoculation and culture is the 4th day
  • Perform cell counting add 50ml of serum-free cell culture medium into the cell culture flask, and put it back into the cell culture incubator for culturing.
  • the growth condition is good, take a sample for cell counting, when the viable cell density is not lower than 1 ⁇ 106 cells/ml, mix the cell culture medium with 750ml serum-free cell culture medium Pour into a cell culture bag and place it back in the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the original cell solution at a concentration of 6 ⁇ 10 7 cells/ml in physiological saline for injection containing 1.5% human serum albumin.
  • Patient 3 stroke patient, age: 62 years old, sex: male.
  • the serum-free cell culture medium is: KBM 581 (CORNING) serum-free cell culture medium (the concentration of IL-2 in the culture medium is 400IU/ml, pH 7.2-7.4)
  • the supernatant was poured off, the cell pellet was shaken off, resuspended with 50ml of sodium chloride injection, and centrifuged at 1200rpm for 10min. After the centrifugation was completed, the supernatant was poured off, the cell pellet was shaken off, and 5ml of serum-free cell culture medium was added, after mixing, 10 ⁇ l of the cell suspension was taken for cell counting. After the cells were resuspended in serum-free cell culture medium at a concentration of 0.3 ⁇ 10 6 cells/ml, they were inoculated into 225 cm 2 cell culture flasks coated with anti-human CD3 antibody. A sample is taken, checked for sterility and labeled, and placed in a cell incubator for incubation.
  • the culture conditions in the incubator are: 37° C., 5% CO 2 .
  • the cell culture bottle is taken out from the incubator; the cell morphology is observed under a microscope: the growth condition is good. Perform cell counting, add 50ml of serum-free cell culture medium into the cell culture flask, and put it back into the cell culture incubator for culturing.
  • the growth condition is good, take a sample for cell counting, when the viable cell density is not lower than 1 ⁇ 106 cells/ml, mix the cell culture medium with 750ml serum-free cell culture medium Pour into a cell culture bag and place it back in the cell culture incubator for cultivation.
  • Activated and expanded lymphocytes are obtained by resuspending the cell stock solution at a concentration of 8 ⁇ 10 7 /ml in physiological saline for injection containing 1% human serum albumin.
  • each PBMC is divided into three equal parts, and then inoculated in 3 kinds of serum-free medium respectively, cultured in the cell culture bottle coated with activated antibody, every 4- Cell counting was carried out every 6 days and the corresponding cell culture medium was supplemented. After 12 days, the culture was terminated and cell counting, viability, phenotype detection, and killing activity detection were performed.
  • the serum-free medium TexMACS GMP Medium was used to culture the cells. After 6-8 days of culture, the cell expansion was slow, which could not meet the quality requirements of lymphocyte expansion in vitro.
  • Example 1 For the preparation of the lymphocyte expansion and activation stock solution. Count the cells using a cytometer to determine the expansion ratio. Expanded cell viability was obtained by trypan blue staining and cell counting. The phenotype detection of the expanded cells was detected by flow cytometry using FITC-labeled CD3 antibody and APC-labeled CD8 antibody. The results are shown in Table 4.
  • Samples were prepared according to Example 1, and intravenous injection was given back to the cerebellar ataxia patient 1 in Table 4.
  • the treatment plan in the first course of treatment, the patient received 2 intravenous infusions, once every two weeks; in the second course of treatment, the patient received 2 The first intravenous infusion, once every three weeks; the third course of treatment, three times of intravenous infusion, once every four weeks.
  • the patient self-perceived that all aspects of the body have improved significantly, and there is a tendency to accelerate the recovery of the body.
  • the improvement in walking is particularly obvious: the dizziness is significantly reduced, the joints are more flexible, the walking is more natural, and the gait is more relaxed.
  • the progress of physical rehabilitation has reached a new level of breakthrough: first, the dizziness is lighter, the mind is clearer, and the body control is enhanced; second, the whole body is more coordinated; third, hip joints, knee joints and other joints The parts are much more flexible, the lower limbs are obviously relaxed, and the walking has made great progress, and the walking has become more comfortable and the gait is consciously flexible and relaxed.
  • the progress of physical rehabilitation has reached a breakthrough new level: first, the mind is clearer and body control is enhanced; second, the whole body is more coordinated; third, the joints such as hip joints and knee joints are flexible, and the lower limbs are obviously Relaxed, good walking self-control, walking has become comfortable and the gait is consciously flexible and relaxed.
  • Peripheral blood was taken according to the scheme of Example 1 for lymphocyte expansion and culture, and the composition was prepared, and then intravenously injected and reinfused by itself.
  • the treatment plan in the first course of treatment, the patient received 2 intravenous infusions, once every two weeks; In the course of treatment, the patient received 2 intravenous infusions, once every three weeks; in the third course of treatment, the patient received 3 intravenous infusions, once every four weeks. After one month of treatment, all the eczema disappeared, and after three months, the skin was as perfect as before.
  • Peripheral blood was taken according to the scheme of Example 1 for lymphocyte expansion and culture, and the composition was prepared, and then intravenously injected and reinfused by itself.
  • the treatment plan in the first course of treatment, the patient received 2 intravenous infusions, once every two weeks; In the course of treatment, the patient received 2 intravenous infusions, once every three weeks; in the third course of treatment, the patient received 3 intravenous infusions, once every four weeks.
  • Peripheral blood was taken according to the scheme of Example 1 for lymphocyte expansion and culture, and the composition was prepared, and then intravenously injected and reinfused by itself.
  • the treatment plan in the first course of treatment, the patient received 2 intravenous infusions, once every two weeks; In the course of treatment, the patient received 2 intravenous infusions, once every three weeks; in the third course of treatment, the patient received 3 intravenous infusions, once every four weeks. After 3 courses of treatment, the state of an illness basically recovered.
  • menopausal syndrome patients menopausal for half a year, insomnia, physical fatigue, the hospital diagnosed menopausal symptoms.
  • Peripheral blood was taken according to the scheme of Example 1 for lymphocyte expansion and culture, and the composition was prepared, and then intravenously injected and reinfused by itself.
  • the treatment plan in the first course of treatment, the patient received 2 intravenous infusions, once every two weeks; In the course of treatment, the patient received 2 intravenous infusions, once every three weeks; in the third course of treatment, the patient received 3 intravenous infusions, once every four weeks. After 4 courses of treatment, the symptoms of discomfort disappeared.

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Abstract

本发明涉及一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：（1）将自体外周全血离心提取获得的外周血单个核细胞（PBMC）与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合；（2）将步骤（1）制得的培养活化淋巴细胞置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物；（3）在步骤（2）传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，扩增培养代数为1-5代；（4）离心，洗涤，收集活化扩增的淋巴细胞，即得。本发明制得的活化扩增淋巴细胞用于防治神经科疾病患者，利于改善并增强患者的身体控制力且使其头脑更清醒，显著减轻小脑共济失调患者的头晕、行动不便等症状，显著提高了患者的生活质量并加速患者康复，显著提高免疫细胞治疗的靶向性和精准性，并显著提高患者的依从性。

Description

一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途 技术领域
本发明涉及生物医药领域，具体涉及一种高效扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途。
背景技术
脑卒中(cerebral stroke)是一种急性脑血管疾病，因脑部血管突然破裂或因血管阻塞导致血液不能流入大脑而引起的脑组织缺血性损伤，并具有发病率高、死亡率高和致残率高等特点。颈内动脉及椎动脉闭塞和狭窄可引起缺血性脑卒中，严重者可引起死亡。
脑外伤多由外物造成的头脑部外伤伤害，并常引起不同程度的永久性功能障碍。不同区域的脑损害可引起包括运动、感觉、言语、视觉、听觉等区域异常局灶性症状。弥散性脑损害甚至影响记忆、睡眠或导致意识模糊和昏迷。
神经系统退行性病变是由于脑或脊髓的神经元或胶质细胞的丢失而导致的慢性进行性中枢神经组织退变，包括帕金森病、阿尔兹海默病、亨廷顿病、肌萎缩侧索硬化症、多发性硬化等。目前，我国帕金森患者超过300万，渐冻症患者超过20万，老年痴呆患者超过千万，随着老年社会的加剧膨大，存在高发病率和高普遍性，状况令人堪忧。
共济失调是指小脑、本体感觉以及前庭功能障碍导致的运动笨拙和不协调，累及躯干、四肢和咽喉肌时可引起身体平衡、姿势、步态及言语障碍。临床上分为小脑性共济失调、大脑性共济失调、感觉性共济失调及前庭性共济失调。共济失调是多种疾病的综合表现，需要研究其潜在的病因学。小脑性共济失调是神经科患者的常见表现，病因多样。小脑变性可能病程很长且缓慢进展，但由梗死、水肿或出血导致的急性小脑肿胀可具有快速且严重的不利影响，属于神经科急症。共济失调的病因复杂，分子学、基因学和影像学诊断技术的发展为正确诊断各种类型共济失调提供更多依据，但由于诊查时机和普及诊断手段的限制，临床上仍有部分患者的发病机制尚未明确。由于患者个体差异的影响，临床的药物治疗手段缺乏足够的循证医学依据，疗效也难以肯定。研究开发更为安全有效的共济失调治疗药物以满足尚未满足的临床需求。
周围神经损伤主要由创伤、肿瘤及代谢性疾病等因素引起的临床常见病，经常会导致机体受累节段运动、感觉和自主功能的部分或全部丧失，以及出现顽固性神经痛，这严重影响患者的生活质量，并为家庭和社会造成了沉重的负担。目前已开发了多种策略来促进周围神经损伤发生后的修复与再生，但是在细胞大量丢失的情况下，如在严重损伤后很可能需要完全替换细胞，细胞治疗时，植入的细胞可产生神经营养因子、结构性和黏附性细胞外基质分子等，为周围神经轴突再生和功能回路重建创造了良好的微环境，从而刺激和支持近端残端的轴突再生，是修复长段周围神经损伤的一种有前景的治疗策略。用 于周围神经损伤修复的细胞类型主要包括成熟体细胞如许旺细胞、嗅鞘细胞，干细胞如胚胎干细胞、神经干细胞、间充质干细胞、成肌细胞和诱导多能干细胞等。
世界卫生组织2017年发布的《全球糖尿病报告》，在全球范围内，糖尿病的患病人数都在不断增加。且随着生活水平的提高和社会竞争压力的加剧使得发病率还在呈现上升和年轻化的趋势。我国糖尿病患者人数为1.29亿，90％以上为2型糖尿病，2型糖尿病发病机制复杂，其多种并发症导致了糖尿病患者致残致死等严重后果。糖尿病患者免疫力往往比较弱，容易患有各种疾病：糖尿病患者糖代谢紊乱，高血糖和组织细胞内含糖增高、丙酮酸积聚，利于病菌的生长繁殖；脂肪代谢障碍，甘油三酯增多，为病菌提供了丰富营养；白蛋白形成减少，体内抗体和补体水平下降，免疫力降低，血液粘度增加，微循环障碍，影响血液与组织间氧交换，导致机体抵抗力降低。糖尿病患者代谢紊乱导致肝脏受损，转化维生素A功能下降，致使呼吸道黏膜上皮完整性受损，防御功能下降，易遭受病菌感染。糖尿病患者或其亲属常伴有其它自身免疫性疾病，如弥漫性甲状腺肿伴甲亢、慢性淋巴性甲状腺炎、重症肌无力、恶性贫血。II型糖尿病老年患者中，恶性肿瘤发生率明显升高，患者体内的胰岛素抵抗会导致高胰岛素血症，而胰岛素有促进恶性肿瘤生长的作用。
鼻腔容易受到细菌、病毒、吸入污染物、灰尘、有害物等侵袭，并引发过敏性鼻炎。过敏性鼻炎(又称为变应性鼻炎，Anaphylactic Rhinitis，AR)是一种鼻黏膜非感染性炎性疾病，过敏原在皮肤黏膜、 鼻黏膜等部位亲密接触，并在肥大细胞表面IGE受体“桥接”引起肥大细胞脱颗粒，使组胺、白三烯、缓激肽等炎症因子释放于局部皮肤、黏膜或血管，引起局部毛细血管扩张、血管通透性增加，平滑肌收缩和腺体分泌增多，症状包括喷嚏、清涕、鼻塞、鼻痒等，且易导致鼻腔菌群紊乱，滋生有害菌。WHO调查数据显示，全球过敏性鼻炎患病率高达11％，而中国人群过敏性鼻炎患病率为10％-15％，过敏性鼻炎患者高达2.4亿。为此，临床急需安全有效防治过敏性鼻炎症状的药物组合物。
湿疹是一种常见的过敏性疾病，容易反复发作，发病人群不分年龄、性别和地域。湿疹产生的原因可能与机体的内在因素、外部环境因素、社会心理因素等都有关系。当机体的免疫失衡后，处于“免疫激活”状态，则会导致过敏反应的出现。人体内的部分免疫细胞战斗士兵头脑发热，不分敌我，抗体、补体等超常规武器开火，开始攻击自己的人民。
荨麻疹俗称风疹块。皮肤、黏膜小血管扩张及渗透性增加而出现的一种局限性水肿反应，通常在2～24小时内消退，但反复发生新的皮疹。病程迁延数日至数月。临床上较为常见。
女性更年期综合症是女性卵巢功能逐渐衰退至完全消失的过渡期，由于生理和心理改变而出现的一系列临床症状，常见有烘热汗出、烦躁易怒、心悸失眠或忧郁健忘等。根据临床表现，本病属于中医学的“绝经前后诸证”的范畴。干细胞能够定向分化出足够的卵细胞，补充卵细胞的数量，刺激雌激素的分泌，维护卵巢正常的形态和功能。 应用干细胞治疗卵巢衰老可以延缓女性更年期的到来，绝经期延后，心理年龄提高，靶向作用，刺激体内荷尔蒙分泌雌激素，提高体内雌激素水平，让女性更年期出现的众多问题得到改善，并较终推迟更年期的到来。
截至目前，临床观察到了大量的免疫细胞治疗、或免疫细胞治疗与手术、放疗、化疗、靶向治疗、免疫激活剂治疗等治疗手段联合使用，使病人治疗获益的病例，使得免疫细胞在免疫治疗中获得了长久的生命力，备受广泛重视并得到广泛应用。
无血清细胞培养扩增活化的淋巴细胞(Expanded Activated Lymphocytes)系由患者自体外周血中的单个核细胞制备而成，主要成分为扩增活化的T淋巴细胞(CD3+细胞)，以CD8+杀伤性T淋巴细胞为主要功能细胞，患者单次回输的CD8+T细胞平均数量为4.70±1.47×10 9个，并在临床实践中显示了很好地治疗活性。现有技术公开了无血清培养扩增活化淋巴细胞的方法，但需优化外周血单个核细胞(PBMC)的提取分离、淋巴细胞活化剂的种类及配比、无血清培养基等，以改善该方法制得的淋巴细胞扩增倍数和细胞活率及生物学活性，满足临床细胞质量稳定可控和安全有效的治疗需求。
发明内容
本发明的目的在于提供一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：
(1)将自体外周全血离心提取获得的外周血单个核细胞(PBMC) 与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，其中，所述外周血单个核细胞在无血清培养基中培养活化的起始密度为(0.2-1.6)×10 6个/ml,细胞活化培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述淋巴细胞活化剂选自抗人CD2抗体、抗人CD3抗体、抗人CD28抗体、植物血凝素(PHA)的任一种或其组合或将其固化在载体上的含抗体载体，所述无血清培养基选自KBM 581、GT-T551H3的任一种或其组合；
(2)将步骤(1)制得的培养活化淋巴细胞按照细胞密度为(0.5-5)×10 6个/ml置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物，其中，传代培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551H3的任一种或其组合，传代培养代数为1-5代；
(3)在步骤(2)传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，其中，扩增培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551H3的任一种或其组合，扩增培养代数为1-5代；
(4)离心，洗涤，收集活化扩增的淋巴细胞，即得。
本发明的优选技术方案中，所述生物学样品在无血清培养基中培养活化的起始密度为(0.2-1)×10 6个/ml。
本发明的优选技术方案中，所述淋巴细胞活化剂为抗人CD3抗体，优选为2.5μg/ml-5μg/ml，体积为8-15ml，更优选为2.5μ g/ml-3.8μg/ml，体积为10-13ml。
本发明的优选技术方案中，所述全血离心提取外周血单个核细胞的方法包括下述步骤：在抗凝处理的全血中加入分离介质和稀释剂，搅拌，混匀，将其加至分离液中，离心1000-3000rpm*10-40min，收集分界面间细胞层，加入洗涤液，离心，洗涤，收集细胞，即得，其中，所述分离介质选自羟乙基淀粉40氯化钠注射液(HES)、Percoll、Ficoll-Paque PLUS的任一种或其组合，全血：稀释剂的体积比为1：1-2，所述稀释剂选自氯化钠注射液、Hank’s缓冲液、Lactated Ringer’s溶液、Dulbecco's磷酸盐缓冲液的任一种或其组合，所述分离液选自羟乙基淀粉40氯化钠注射液(HES)、Ficoll、Lymphoprep、Lymphocyte Separation Media、Cell Separation Media的任一种或其组合，所述分离液的渗透压为300mOsmol/kg-360mOsmol/kg，所述洗涤液选自0.1％人血白蛋白氯化钠注射液、Dulbecco's磷酸盐缓冲液、氯化钠注射液的任一种或其组合。
本发明的优选技术方案中，离心条件为(1500-2500rpm)*(15-30min)，优选为(2000-2500rpm)*(20-25min)。
本发明的优选技术方案中，洗涤为离心洗涤，洗涤条件为(500-2000rpm)*(5-20min)，离心洗涤1-5次,优选为(1000-1800rpm)*(10-15min)，离心洗涤2-3次。
本发明的优选技术方案中，步骤(2)中传代培养的细胞密度为(1-4)×10 6个/ml，传代培养体系添加无血清培养基后pH 7.00-7.80。
本发明的优选技术方案中，步骤(2)中,传代培养的细胞密度为 (2-3)×10 6个/ml，传代培养体系添加无血清培养基后pH 7.02-7.76。
本发明的优选技术方案中，所述分离液的渗透压为310-350mOsmol/kg，优选为316-347mOsmol/kg。
本发明的优选技术方案中，所述传代培养代数为2-3代。
本发明的优选技术方案中，步骤(3)中，当传代培养细胞密度增长至培养活化细胞密度的1-10倍时开始扩增培养，将步骤(2)传代培养获得的淋巴细胞培养物加入其体积共6-8倍的无血清培养基中扩增培养，所述扩增培养体系pH 6.80-7.80。
本发明的优选技术方案中，步骤(3)中，当传代培养细胞密度增长至培养活化细胞密度的1.2-3倍时开始扩增培养，所述扩增培养体系pH 6.88-7.70。
本发明的优选技术方案中，所述扩增培养代数为2-3代。
本发明的优选技术方案中，步骤(4)中的离心条件为(1000-3000rpm)*(1-10min)，优选为(1500-2800rpm)*(2-8min)，更优选为(2000-2500rpm)*(5-6min)。
本发明的优选技术方案中，所述无血清培养基中任选地添加细胞因子IL-2 300-600IU/ml，优选为400-500IU/ml。
本发明的优选技术方案中，所述无血清培养基中pH为6.9-7.9，优选为7.2-7.4。
本发明的优选技术方案中，所述培养设备选自培养箱、摇床、生物反应器的任一种。
本发明的优选技术方案中，活化扩增淋巴细胞扩增倍数≥900倍， 优选≥1000倍，更优选≥1100倍。
本发明的优选技术方案中，活化扩增淋巴细胞活率≥95％，优选≥98％。
本发明的优选技术方案中，活化扩增淋巴细胞中的CD8+T细胞数量≥1×10 9个、优选为1×10 9-2×10 10个，更优选为4-9.5×10 9个。
本发明的优选技术方案中，活化扩增后的淋巴细胞生物学活性KT 50≤8.5，优选为KT 50≤4，更优选为KT 50≤0.7141。
本发明的另一目的在于提供一种含有活化扩增淋巴细胞原液的药物组合物，所述组合物由(1-20)×10 7个/ml的活化扩增淋巴细胞、0.5-2％的人血白蛋白和注射用生理盐水组成，活化扩增淋巴细胞活率≥95％，其中，活化扩增淋巴细胞中的CD8+T细胞为(1-20)×10 7个/ml，所述药物组合物中不含防腐剂和抗生素。
本发明的优选技术方案中，所述组合物由(2-18)×10 9个/ml的活化扩增淋巴细胞浓度、1-1.5％人血白蛋白浓度和注射用生理盐水组成，活化扩增淋巴细胞活率≥98％，活化扩增淋巴细胞中的CD8+T细胞为1×10 9-2×10 10个。
本发明的优选技术方案中，生物学样品的细胞扩增倍数≥900倍，优选为≥1000倍，更优选为≥1100倍。
本发明的优选技术方案中，活化扩增淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
本发明的优选技术方案中，患者单次回输活化扩增淋巴细胞总数 ≤2×10 10个，有效期内细胞活率≥85％。
本发明的优选技术方案中，组合物于15-25℃保存12h内的活化扩增淋巴细胞活率≥85％。
本发明的另一目的在于提供一种含有活化扩增淋巴细胞原液的药物组合物的制备方法，将活化扩增淋巴细胞原液重悬于含有人血白蛋白的注射用生理盐水中。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞用于免疫治疗的方法，包括下述步骤：
(1)从个体获得含有淋巴细胞的生物学样品；
(2)采用本发明的制备方法活化、传代、扩增所述淋巴细胞，获得活化扩增淋巴细胞；
(3)将所述活化扩增淋巴细胞或其药物组合物回输给所述个体。
本发明的另一目的在于提供本发明的活化扩增淋巴细胞用于免疫治疗的给药方案，包括下述方案：
(1)第一疗程，患者接受1-5次静脉输注，每两周1次；
(2)第二疗程，患者接受1-5次静脉输注，每三周1次；
根据需要增加疗程，增加疗程输注间隔时间延长。
本发明优选的技术方案中，包括下述方案：
(1)第一疗程，患者接受2-4次静脉输注，每两周1次；
(2)第二疗程，患者接受2-4次静脉输注，每三周1次；
(3)第三疗程，患者接受2-4次静脉输注，每四周1次。
本发明的优选技术方案中，每次静脉输注的活化扩增淋巴细胞数≥5x10 8个淋巴细胞，优选为≥(1-20)x10 9个淋巴细胞。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备防治神经科疾病患者的药物中的应用，所述神经科疾病患者需要增强免疫力、神经修复和/或神经营养，以修复和/或营养神经性创伤、缺血缺氧导致的神经损伤。
本发明优选的技术方案中，所述神经科疾病选自共济失调、痴呆症、癫痫病、中风、脑卒中后遗症、帕金森氏病、神经退行性病变、手颤症、周围神经和肌肉疾病、头痛、睡眠障碍、头晕、神经系统炎症的任一种或其并发症。
本发明的优选技术方案中，所述共济失调选自小脑性共济失调、大脑性共济失调、感觉性共济失调、前庭性共济失调的任一种或其组合。
本发明的优选技术方案中，所述共济失调选自获得性共济失调、遗传性共济失调、非遗传性退行性共济失调的任一种。
本发明的另一目的在于提供活化扩增淋巴细胞与康复训练联合用于防治神经科疾病的应用。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备治疗过敏性疾病患者的药物中的应用。
本发明的优选技术方案中，所述过敏性疾病包括鼻炎、湿疹、荨麻疹中的任一种。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备治疗更年期综合症患者的药物中的应用。
本发明的另一目的在于提供活化扩增淋巴细胞用于制备治疗糖尿病患者的药物中的应用。
除非另有说明，本发明采用扩增活化的淋巴细胞生物学活性(RTCA，以效靶比40:1为例)来表征活化扩增淋巴细胞的治疗活性。将消化好的靶细胞(如HepG2等)按照每孔加入100μl的密度为2×10 5cells/ml靶细胞提前铺板，并设置好效靶比参数(如效靶比为40:1)进行基线测量。在靶细胞铺板检测16-24h后，按照设定的效靶比加入特定密度(每孔加入50μl密度为1.6×10 7cell/ml)的效应细胞(即扩增活化的淋巴细胞)后，实时、动态、定量跟踪细胞形态和增殖分化等数据，即活细胞与检测板中的微电极接触产生电阻抗的改变。当在含有贴壁细胞的孔中加入效应细胞后，贴壁生长在微电极表面的细胞引起贴壁电极界面阻抗的改变，这种改变与细胞指数(Cell Index)的改变呈正相关性。仪器专用软件将电阻信号转化为特定的细胞指数，软件实时监测产生的细胞指数曲线，以展现细胞在不同时间段内的黏附、伸展、生长、死亡等状态，通过分析细胞指数的改变，即可计算出效应细胞对靶细胞(如HepG2细胞)的杀伤效率，通过杀伤效率曲线，计算出KT 50(即效应细胞杀伤50％靶细胞所用时间)，并以KT 50值作为判定效应细胞(即活化扩增淋巴细胞)生物学活性的 指标。KT 50值越小，说明杀伤效率越高，生物学活性越高。反之，则生物学活性越低。
除非另有说明，本发明涉及液体与液体之间的百分比时，所述的百分比为体积/体积百分比；本发明涉及液体与固体之间的百分比时，所述百分比为体积/重量百分比；本发明涉及固体与液体之间的百分比时，所述百分比为重量/体积百分比；其余为重量/重量百分比。
与现有技术相比，本发明具有下述有益技术效果：
1、本发明制得的活化扩增淋巴细胞具有细胞扩增效率高、细胞活率高、细胞活性稳定、具有较长的有效期、安全有效、副作用小等优点，有效解决了个体化免疫细胞治疗的关键问题，一是使用具有稳定细胞培养效率的无血清培养体系，持续培养12天而始终保持细胞的杀伤活性和扩增能力，且避免了使用血清可能导致的病源微生物污染及其它对细胞生长的不利因素；二是细胞扩增效率高，不需要使用单核细胞采集机，高效扩增1000倍以上，避免了传统免疫细胞治疗需要使用单采机采取大量起始外周血单个核细胞对患者整体免疫细胞系统的破坏作用；三是细胞活性高、稳定性好，细胞可保存12h而不影响其治疗效果；四是解决了个体化产品的标准化质量控制体系、规模化生产的技术问题，获得相对质量均一的细胞产品的技术工艺，显著提高治疗靶向性和治疗精准性及患者的依从性。
2、本发明制得的活化扩增淋巴细胞用于防治神经科疾病患者，所述神经科疾病患者需要增强免疫力、神经修复和/或神经营养，以修复和/或营养神经性创伤、缺血缺氧导致的神经损伤，显著改善患 者的神经传导、关节灵活性与运动协调性，利于改善并增强患者的身体控制力且使其头脑更清醒，显著减轻小脑共济失调患者的头晕、行动不便等症状，显著提高了患者的生活质量并加速患者康复，具有细胞扩增效率高、细胞活率高、细胞稳定性好、疗效显著、恢复快、安全有效、副作用小等优点，显著提高免疫细胞治疗的靶向性和精准性，并显著提高患者的依从性。
3、本发明的淋巴细胞扩增方法具有操作简便、质量可控，适宜工业化生产等优点。
附图说明
图1实施例1第一次传代培养时EAL细胞生长状况(250×镜下观察)
图2实施例1第一次扩增培养时EAL细胞生长状况(250×镜下观察)
图3实施例1第二次扩增培养时EAL细胞生长状况(250×镜下观察)
具体实施方式
以下结合实施例对本发明做进一步的说明，但并不因此将本发明限制在所描述的实施例范围中。
实施例1淋巴细胞活化扩增原液制备
(1)分离外周血单个核细胞(PBMC)并接种活化培养(第0天)
患者1：小脑共济失调，年龄：52岁，性别：男。
所述无血清细胞培养基为：GT-T551H3无血清细胞培养基(培养基中IL-2的浓度为500IU/ml,pH 7.2-7.4)
将抽取的患者全血37ml放入一新250ml离心管中，加入与血样等体积的0.9％氯化钠注射液，混匀后，将其缓慢加到已分装好15ml Ficoll的50ml离心管中(2根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后，倾去上清，振开细胞沉淀后，用50ml 0.9％氯化钠注射液重悬，1200rpm离心10min。离心完成后，倾去上清，振开细胞沉淀后，加5ml无血清细胞培养基，混匀后，取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞浓度为0.8×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中，取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，7.5％CO 2。
(2)第一次传代培养(第3天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第3天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好(见图1)。添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第4天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。添加140ml无血清细胞培养基，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第5天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好(见图2)，取样进行细胞计数，当活细胞密度不低于1.0×10 6个/ml时，将细胞培养液和760ml无血清细胞培养基，倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态(见图3)，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml含0.1％人血白 蛋白的0.9％氯化钠注射液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管，2000rpm离心5分钟。倾去上清，振散沉淀，再用200ml 0.1％人血白蛋白氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后，取样进行细胞计数，2000rpm离心5分钟，倾去上清后，即为活化扩增淋巴细胞原液。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为8×10 7个/ml重悬细胞原液于含有1％人血白蛋白的注射用生理盐水中，即得。
实施例2淋巴细胞活化扩增原液制备
(1)分离PBMC并接种活化培养(第0天)
患者2：小脑共济失调，年龄：65岁，性别：男。
所述无血清细胞培养基为：KBM 581无血清细胞培养基(培养基中IL-2的浓度为500IU/ml,pH 7.2-7.4)
将63ml全血放入一新250ml离心管中，加入与血样等体积的氯化钠注射液，混匀后缓慢加到已分装好15ml Ficoll的50ml离心管中(4根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后用50ml氯化钠注射液重悬，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后加5ml无血清细胞培养基，混匀后取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞 浓度为0.2×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中。取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，7.5％CO 2。
(2)第一次传代培养(第4天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第4天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第5天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加140ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第6天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好，取样进行细胞计数，当活细胞密度不低于1×10 6个/ml时，将细胞培养液和750ml无血清细胞培养基倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培 养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml 0.9％氯化钠注射液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管，2000rpm离心5分钟。倾去上清振散沉淀，然后用200ml氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后取样进行细胞计数，然后2000rpm离心5分钟，倾去上清后即为活化扩增淋巴细胞原液。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为6×10 7个/ml重悬细胞原液于含有1.5％人血白蛋白的注射用生理盐水中，即得。
实施例3淋巴细胞活化扩增原液制备
(1)分离PBMC并接种活化培养(第0天)
患者3：中风患者，年龄：62岁，性别：男。
所述无血清细胞培养基为：KBM 581(CORNING)无血清细胞培养基(培养基中IL-2的浓度为400IU/ml,pH 7.2-7.4)
将抽取患者的58ml全血放入一新250ml离心管中，加入与血样等体积的0.9％氯化钠注射液，混匀后缓慢加到已分装好15ml Ficoll的50ml离心管中(4根离心管)，保证分界面清晰，2000rpm离心20min，离心机设置加速7减速0。吸取界面间细胞层到一新50ml离心管，用0.9％氯化钠注射液1:1混匀，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后用50ml氯化钠注射液重悬，1200rpm离心10min。离心完成后倾去上清，振开细胞沉淀后加5ml无血清细胞培养基，混匀后取10μl细胞悬液进行细胞计数。用无血清细胞培养基重悬细胞浓度为0.3×10 6个/ml后，接种到包被有抗人CD3抗体的225cm 2细胞培养瓶中。取样，进行无菌检查并进行标记，并将其放入细胞培养箱进行培养。
淋巴细胞活化扩增原液制备过程中，培养箱培养条件：37℃，5％CO 2。
(2)第一次传代培养(第3天)
当培养基颜色变浅变黄时(以采血当日为第0天计，接种培养后第3天)，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加50ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(3)第二次传代培养(第4天)
当培养基颜色变浅变黄时，从培养箱取出细胞培养瓶；显微镜下观察细胞形态：生长状况良好。进行细胞计数，添加140ml无血清细胞培养基倒入细胞培养瓶中，将其放回细胞培养箱进行培养。
(4)第一次扩增培养(第6天)
从培养箱取出细胞培养瓶，显微镜下观察细胞形态：生长状况良好，取样进行细胞计数，当活细胞密度不低于1×10 6个/ml时，将细胞培养液和750ml无血清细胞培养基倒入细胞培养袋中，将其放回细胞培养箱进行培养。
(5)第二次扩增培养(第8天)
当培养液颜色偏黄时，取一新细胞培养袋至生物安全柜，通过注射器套筒倒入1000ml无血清细胞培养基(袋A)，封死管口。从培养箱取出含有培养中细胞的细胞培养袋(袋B)，通过无菌接合机将两袋连通，首先使袋A中液体全部流入袋B中，混匀后再放回1000ml至袋A，两袋中液体量均为1000ml。封死管口，抽取样品进行无菌检查。显微镜下观察细胞形态，将两袋细胞放回培养箱中进行培养。
(6)纯化、离心浓缩收集细胞制成细胞原液(第12天)
两个细胞培养袋后第4天，从培养箱中取出一细胞培养袋至生物安全柜，将细胞悬液均分到4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀。再从培养箱中取出同一批次另一细胞培养袋至生物安全柜，将细胞悬液均分到原来的4个250ml离心管中，2000rpm离心5分钟。倾去上清振散沉淀，用250ml 0.9％氯化钠注射液将一个管中细胞分三次冲洗合并到另一个管中，四管合并至两管， 2000rpm离心5分钟。倾去上清，振散沉淀，再用200ml氯化钠注射液将两个管中细胞分三次冲洗合并到一个管中，混匀后取样进行细胞计数，2000rpm离心5分钟，倾去上清后即为扩增活化淋巴细胞原液。
(7)组合物的制备
活化扩增淋巴细胞按照浓度为8×10 7个/ml重悬细胞原液于含有1％人血白蛋白的注射用生理盐水中，即得。
实施例4无血清培养基的筛选
采用与实施例1相同的活化扩增工艺，体外比较研究三种无血清培养基KBM 581(CORNING)、GT-T551H3(TAKARA)、TexMACS GMP Medium(Miltentyi biotec)对EAL细胞制备的影响。
采集3个正常人外周血分离PBMC，进行细胞计数和表型检测，每份PBMC三等分后分别用3种无血清培养基，接种于已包被活化抗体细胞培养瓶进行培养，每4-6天进行一次细胞计数并补充相应细胞培养基，12天后结束培养并进行细胞计数、活率、表型检测、杀伤活性检测。
使用无血清培养基TexMACS GMP Medium培养细胞，细胞在培养6～8天后细胞扩增缓慢，无法满足淋巴细胞体外扩增质量要求。
表1
Figure PCTCN2022142961-appb-000001
Figure PCTCN2022142961-appb-000002
表2
Figure PCTCN2022142961-appb-000003
表3
Figure PCTCN2022142961-appb-000004
实施例5活化扩增淋巴细胞原液制备
(1)采集了共计18例患者的外周血，不同性别、年龄和类型均有分布。患者基本信息见表4。淋巴细胞扩增活化原液的制备参考实施例1。使用细胞计数板对细胞进行计数，以测定扩增比例。通过台盼蓝染色以及细胞计数获得扩增的细胞活率。通过流式细胞术检测扩增细胞的表型检测使用FITC标记的CD3抗体、APC标记的CD8抗体测定，结果见表4。
表4
Figure PCTCN2022142961-appb-000005
试验例1小脑共济失调治疗研究
按照实施例1制备样品，静脉注射回输给表4的小脑共济失调患者1，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。
治疗前：头晕目眩；日常生活(如持物、穿衣、进食、坐起等)不能完全自理，且双侧肢体活动失衡、站立行走不能。
治疗后随访结果：
第一次治疗后：患者自我感知身体各方面都有显著进步，且存在身体加速康复的趋势，走路方面的进步表现特别明显：头晕显著减轻，关节更灵活，走路更自然，步履更轻松。
第二次治疗后：患者静止时不再感觉头晕，走路运动中的头晕己显著轻微。走路时，觉得双脚力量在逐步平衡，髋关节、膝关节、裸关节更灵活且具弹性。身体自控能力增强，行走显得自我控制。患者自觉进步很大。
第三次治疗后：身体康复进步达到了一个突破性的新高度：一是头晕更轻徽，头脑更清醒，身体控制力增强；二是整个身体更协调；三是髋关节、膝关节等关节部位灵活很多，下肢明显轻松，走路上进步非常大，行走已显自如且步履自觉灵活轻松。
第六次治疗后：身体康复进步达到了一个突破性的新高度：一是头脑更清醒，身体控制力增强；二是整个身体更协调；三是髋关节、膝关节等关节部位灵活，下肢明显轻松，走路自我控制良好，行走已显自如且步履自觉灵活轻松。
另有2名小脑共济失调患者治疗效果明显。
试验例2中风治疗研究
3名中风患者，患者在治疗前已经患有大于2年的大中风。按实施例1的方案取外周血进行淋巴细胞扩增培养，制备组合物，然后自身静脉注射回输，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。治疗2个疗程后，3名患者的认知功能，步态速度和Barthel指数性能显著改善。
试验例3湿疹治疗研究
3名湿疹患者，皮肤湿疹，睡眠不好，精力下降。多次治疗未愈，皮肤上反复多次出现湿疹，MISS免疫评分为-5分，淋巴细胞亚群出现了严重下降和失衡，CD4+T细胞水平代偿激活。
按实施例1的方案取外周血进行淋巴细胞扩增培养，制备组合物，然后自身静脉注射回输，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。治疗一个月后湿疹全部消失，三个月后皮肤完美如初。
试验例4鼻炎治疗研究
3名鼻炎患者，季节性过敏性鼻炎，每次秋季发作，每次持续1-2个月，伴有鼻塞、鼻涕、有眼泪、头晕等症状。
按实施例1的方案取外周血进行淋巴细胞扩增培养，制备组合物，然后自身静脉注射回输，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。
治疗后2个疗程后随访结果：患者自我感知身体各方面都有显著进步，且鼻塞、鼻涕症状有所减轻。
试验例5荨麻疹治疗研究
3名慢性荨麻疹患者，服用氯雷他定、左西替利抗过敏药及外擦制剂但并无好转。脸上很多红色风团，久久不能消退，奇痒无比，彻夜难眠，精神状态很差，并且严重影响生活及社交。
按实施例1的方案取外周血进行淋巴细胞扩增培养，制备组合物，然后自身静脉注射回输，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。治疗3个疗程后，病情基本痊愈。
试验例6更年期综合症治疗研究
3名更年期综合症患者，绝经半年，失眠、身体乏力，医院诊断更年期症状。
按实施例1的方案取外周血进行淋巴细胞扩增培养，制备组合物，然后自身静脉注射回输，治疗方案：第一疗程，患者接受2次静脉输注，每两周1次；第二疗程，患者接受2次静脉输注，每三周1次；第三疗程，接受3次静脉输注，每四周1次。治疗4个疗程后，不适症状消失。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明权利要求保护的范围。

Claims (20)

  1. 一种活化扩增淋巴细胞原液的制备方法，包括下述步骤：
    (1)将自体外周全血离心提取获得的外周血单个核细胞(PBMC)与淋巴细胞活化剂置于无血清培养基中共培养，完成淋巴细胞的培养活化，其中，所述外周血单个核细胞在无血清培养基中培养活化的起始密度为(0.2-1.6)×10 6个/ml,细胞活化培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述淋巴细胞活化剂选自抗人CD2抗体、抗人CD3抗体、抗人CD28抗体、植物血凝素(PHA)的任一种或其组合或将其固化在载体上的含抗体载体，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合；
    (2)将步骤(1)制得的培养活化淋巴细胞按照细胞密度为(0.5-5)×106个/ml置于无血清培养基中，进行传代培养，制得淋巴细胞活化培养物，其中，传代培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合，传代培养代数为1-5代；
    (3)在步骤(2)传代培养获得的淋巴细胞活化培养物中加入其体积5-10倍的无血清培养基，进行扩增培养，其中，扩增培养温度为37.0℃±1.0℃，活化培养环境中含7.5％±1.0％CO 2，所述无血清培养基选自KBM 581、GT-T551 H3的任一种或其组合，扩增培养代数为1-5代；
    (4)离心，洗涤，收集活化扩增的淋巴细胞，即得。
  2. 如权利要求1所述的方法，其特征在于，所述全血离心提取外 周血单个核细胞的方法包括下述步骤：在抗凝处理的全血中加入分离介质和稀释剂，搅拌，混匀，将其加至分离液中，离心1000-3000rpm*10-40min，收集分界面间细胞层，加入洗涤液，离心，洗涤，收集细胞，即得，其中，所述分离介质选自羟乙基淀粉40氯化钠注射液(HES)、Percoll、Ficoll-Paque PLUS的任一种或其组合，全血：稀释剂的体积比为1：1-2，所述稀释剂选自氯化钠注射液、Hank’s缓冲液、Lactated Ringer’s溶液、Dulbecco's磷酸盐缓冲液的任一种或其组合，所述分离液选自羟乙基淀粉40氯化钠注射液(HES)、Ficoll、Lymphoprep、Lymphocyte Separation Media、Cell Separation Media的任一种或其组合，所述分离液的渗透压为300mOsmol/kg-360mOsmol/kg，所述洗涤液选自0.1％人血白蛋白氯化钠注射液、Dulbecco's磷酸盐缓冲液、氯化钠注射液的任一种或其组合。
  3. 如权利要求1-2任一项所述的方法，其特征在于，离心条件为(1500-2500rpm)*(15-30min)，优选为(2000-2500rpm)*(20-25min)，洗涤为离心洗涤，洗涤条件为(500-2000rpm)*(5-20min)，离心洗涤1-5次,优选为(1000-1800rpm)*(10-15min)，离心洗涤2-3次。
  4. 如权利要求1-3任一项所述的方法，其特征在于，所述无血清培养基中任选地添加细胞因子IL-2 300-600IU/ml，优选为400-500IU/ml。
  5. 如权利要求1-4任一项所述的方法，其特征在于，活化扩增淋 巴细胞扩增倍数≥900倍，优选≥1000倍，更优选≥1100倍。
  6. 如权利要求1-5任一项所述的方法，其特征在于，活化扩增淋巴细胞活率≥95％，优选≥98％。
  7. 如权利要求1-6任一项所述的方法，其特征在于，活化扩增淋巴细胞中的CD8+T细胞数量≥1×10 9个、优选为1×10 9-2×10 10个，更优选为4-9.5×10 9个。
  8. 如权利要求1-7任一项所述的方法，其特征在于，活化扩增后的淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
  9. 一种含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞原液的药物组合物，所述组合物由(1-20)×10 7个/ml的活化扩增淋巴细胞、0.5-2％的人血白蛋白和注射用生理盐水组成，活化扩增淋巴细胞活率≥95％，其中，活化扩增淋巴细胞中的CD8+T细胞为(1-20)×10 7个/ml，所述药物组合物中不含防腐剂和抗生素。
  10. 如权利要求9所述的药物组合物，其特征在于，生物学样品的细胞扩增倍数≥900倍，优选为≥1000倍，更优选为≥1100倍。
  11. 如权利要求9-10任一项所述的药物组合物，其特征在于，活化扩增淋巴细胞生物学活性KT50≤8.5，优选为KT50≤4，更优选为KT50≤0.7141。
  12. 如权利要求9-11任一项所述的药物组合物，其特征在于，患者单次回输活化扩增淋巴细胞总数≤2×10 10个，有效期内细胞活率≥85％。
  13. 如权利要求9-12任一项所述的药物组合物，其特征在于，组合物于15-25℃保存12h内的活化扩增淋巴细胞活率≥85％。
  14. 如权利要求9-13任一项所述的含有活化扩增淋巴细胞原液的药物组合物的制备方法，将活化扩增淋巴细胞原液重悬于含有人血白蛋白的注射用生理盐水中。
  15. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于免疫治疗的方法，包括下述步骤：
    (1)从个体获得含有淋巴细胞的生物学样品；
    (2)采用本发明的制备方法活化、传代、扩增所述淋巴细胞，获得活化扩增淋巴细胞；
    (3)将所述活化扩增淋巴细胞或其药物组合物回输给所述个体。
  16. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于免疫治疗的给药方案，其特征在于，包括下述方案：
    (1)第一疗程，患者接受1-5次静脉输注，每两周1次；
    (2)第二疗程，患者接受1-5次静脉输注，每三周1次；
    根据需要增加疗程，增加疗程输注间隔时间延长。
  17. 如权利要求16所述的给药方案，其特征在于，包括下述方案：
    (1)第一疗程，患者接受2-4次静脉输注，每两周1次；
    (2)第二疗程，患者接受2-4次静脉输注，每三周1次；
    (3)第三疗程，患者接受2-4次静脉输注，每四周1次。
  18. 如权利要求15-17任一项所述的给药方案，其特征在于，每次静脉输注的活化扩增淋巴细胞数≥5x10 8个淋巴细胞，优选为≥ (1-20)x10 9个淋巴细胞。
  19. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于制备防治神经科疾病患者的药物中的应用，所述神经科疾病患者需要增强免疫力、神经修复和/或神经营养，以修复和/或营养神经性创伤、缺血缺氧导致的神经损伤。
  20. 含有如权利要求1-8任一项所述制备方法制备得到的活化扩增淋巴细胞用于制备治疗过敏性疾病患者的药物、制备治疗更年期综合症患者的药物、制备治疗糖尿病患者的药物中的任一种的应用。
PCT/CN2022/142961 2021-12-28 2022-12-28 一种质量稳定可控的扩增活化淋巴细胞的方法及其用于防治神经科疾病中的用途 WO2023125704A1 (zh)

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